Biomarkers of ovarian cancer & molecular genetics of HOSE cell transformation

卵巢癌的生物标志物

• 批准号: 6504969
• 负责人: ANDREW K. GODWIN
• 金额: $ 16.54万
• 依托单位: FOX CHASE CANCER CENTER
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-26 至 2002-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6504969
• 关键词: biomarker; brca gene; gene expression; genetic mapping; human tissue; immunocytochemistry; in situ hybridization; molecular cloning; molecular genetics; neoplastic transformation; northern blottings; ovary neoplasms

DESCRIPTION: (Applicant's Description) The goal of this project is to elucidate the genetic and biological determinants of ovarian cancer, focusing on an in vitro model for ovarian cancer that we have developed (1-4). First, we propose to identify genes that are differentially expressed upon malignant transformation of HOSE cells using a modified suppression subtractive hybridization (SSH) approach. We will use consolidative SSH (CSSH) to identify genes that are either over-expressed or under-represented upon immortalization and malignant transformation of HOSE cells. Second, we propose to identify genes that are differentially expressed in HOSE cells heterozygous for BRCA1 (+/-) using CSSH. We have previously reported that the surface epithelial cells from ovaries of BRCA1 mutant allele carriers have a different phenotype in vitro compared to control surface epithelial cells and these features are present without the inactivation of the remaining wild-type allele of BRCA1. These results suggest that other genetic changes might precede loss of both copies of BRCA1 in the genesis of familial ovarian cancer. We will compare populations of HOSE cells for differentially expressed genes related to their BRCA1 carrier status. Third, we propose to determine the expression pattern of candidate ovarian cancer-causing genes identified by CSSH in benign and borderline ovarian tumors and ovarian carcinomas. To find the subset of genes discovered in our tissue culture model, which are relevant to clinical ovarian tumors, cDNA arrays representing the differentially expressed clones will be created. Reverse transcribed RNAs derived from benign, borderline, early and late stage ovarian tumors will be hybridized to individual arrays and the patterns of expression determined. Clones showing expression patterns consistent with the findings in the model will be evaluated for tissue specific expressions by multiple tissue northern blot analysis. Fourth, we propose to establish the pattern of expression of candidate ovarian cancer-causing genes in preneoplastic lesions by performing in situ hybridization on cancer prone ovaries. We have previously identified a preneoplastic phenotype in the ovaries from women at increased risk for developing ovarian cancer. Once we have confirmed the expression of several genes, in situ hybridization and immunohistochemical approaches will be used to evaluate their RNA and protein expression patterns at potentially the earliest stages of cancer development. Overall, the studies proposed should enable us to (i) determine if the histological phenotype we have previously described is likely to be a true precursor of ovarian cancer, (ii) establish whether other genetic changes precede BRCA1 inactivation in ovarian carcinogenesis, (iii) identify genes that are specifically expressed in the ovarian surface epithelium as opposed to the entire ovary, and (iv) identify early surrogate intermediate endpoint bio- markers of impending ovarian cancer.

产品说明：（申请人的描述）本项目的目标是阐明卵巢癌的遗传和生物学决定因素，重点是我们开发的卵巢癌体外模型（1-4）。 首先，我们建议使用改良的抑制性消减杂交（SSH）方法鉴定HOSE细胞恶性转化后差异表达的基因。 我们将使用巩固SSH（CSSH），以确定基因，无论是过表达或代表性不足的永生化和恶性转化的HOSE细胞。其次，我们建议使用CSSH鉴定BRCA 1（+/-）杂合子HOSE细胞中差异表达的基因。 我们以前曾报道，BRCA 1突变等位基因携带者卵巢的表面上皮细胞在体外具有不同的表型，与对照表面上皮细胞相比，这些特征是存在的，而没有其余的BRCA 1野生型等位基因的失活。 这些结果表明，在家族性卵巢癌的发生中，其他遗传变化可能先于BRCA 1两个拷贝的丢失。 我们将比较HOSE细胞群体中与BRCA 1携带状态相关的差异表达基因。 第三，我们建议确定的候选人卵巢癌的致癌基因的表达模式，在良性和交界性卵巢肿瘤和卵巢癌的CSSH确定。 为了找到在我们的组织培养模型中发现的与临床卵巢肿瘤相关的基因子集，将创建代表差异表达克隆的cDNA阵列。 将来自良性、交界性、早期和晚期卵巢肿瘤的逆转录RNA与单个阵列杂交，并确定表达模式。 将通过多组织北方印迹分析评价显示与模型中的发现一致的表达模式的克隆的组织特异性表达。 第四，我们建议通过对卵巢癌易感性进行原位杂交，建立肿瘤前病变中候选卵巢癌致病基因的表达模式。 我们之前已经在患卵巢癌风险增加的女性的卵巢中发现了肿瘤前表型。 一旦我们确认了几个基因的表达，原位杂交和免疫组织化学方法将被用来评估它们的RNA和蛋白质表达模式在潜在的癌症发展的最早阶段。 总的来说，所提出的研究应该使我们能够（i）确定我们先前描述的组织学表型是否可能是卵巢癌的真正前体，（ii）确定在卵巢癌发生中BRCA 1失活之前是否有其他遗传变化，（iii）鉴定在卵巢表面上皮特异性表达的基因，而不是整个卵巢，和（iv）鉴定即将发生的卵巢癌的早期替代中间终点生物标志物。